Method for treating hypocalcemia

ABSTRACT

A method for treating hypothyroidism by local administration of a neurotoxin, such as a botulinum toxin, to a thyroid, thereby reducing an inhibitory effect upon thyroid hormone secretion. A method for treating hyperthyroidism by local administration of a neurotoxin, such as a botulinum toxin, to a sympathetic ganglion which innervates the thyroid, thereby reducing a stimulatory effect upon thyroid hormone secretion. Methods for treating calcium metabolism disorders by local administration of a neurotoxin to modulate calcitonin secretion are also disclosed.

CROSS REFERENCE

This application is a divisional of Ser. No. 09/504,538, filed Feb. 15,2000.

BACKGROUND

The present invention relates to methods for treating thyroid disorders.In particular the present invention relates to methods for treatingthyroid disorders by administration of a neurotoxin to a patient.

It has been estimated that at least about two hundred million peopleworldwide are afflicted with a thyroid disorder and women are affecteddisproportionaly, as compared to men, by a ratio of about ten to one. Inthe United States, about ten million persons, including ten percent ofall women over age 45, have either overactive or underactive thyroidglands. Bayliss et al., Thyroid Disease The Facts, preface, OxfordUniversity Press (1998).

thyroid Function

The thyroid is an endocrine gland comprised of follicle cells andnon-follicular or C cells. The follicle cells are capable of making twohormones, triiodothyronine (T₃), which contains three iodine atoms andthyroxine (T₄) which contains four. The action of thyroid hormone isconcerned principally with the regulation of metabolic rate by, forexample, increasing energy production and oxygen consumption by mostnormal tissues. Synthesis and release of T₃ and T₄ by thyroid cells ininfluenced by thyroid stimulating hormone (TSH, also calledthyrotrophin) made by the pituitary. The C cells can make calcitoninwhich appears to influence calcium metabolism. Significantly, calcitoninis a potent hypocalcemic agent. Disorders of the thyroid includeautoimmune disorders (such as Graves' disease), thyroiditis(inflammation or infection of the thyroid), and cancer, all of whichconditions can result in hypothyroidism (as can occur in Hashimoto'sthyroiditis) or hyperthyroidism (thyroidtoxicosis, as can occur inGraves' disease). An enlarged thyroid (goiter) can by euthyroid, or asymptom of either hyperthyroidism (thyroidtoxicosis) or hypothyroidism.

Most cases of hyperthyroidism are believed to be due to the action ofthyroid stimulating antibodies upon the thyroid as a whole (Graves'disease, diffuse toxic goiter).

Graves' disease has been estimated to occur in 0.4% of the population ofthe United States, with a lifetime risk of 1%. It is most commonlymanifest in the third or fourth decade of rue and the female to maleratio is about 7:1 to about 10:1. The thyroid abnormalitiescharacteristic of Graves' disease apparently result from the action ofimmunoglobulin of the IgG class on the thyroid. These antibodies may bedirected against components or regions of the plasma membrane thatinclude the receptor for thyroid simulating hormone (TSH) itself Theprincipal destabilizing factor resulting in autoimmune thyroid diseaseappears to be an organ specific defect in suppressor T-lymphocytes.Hyperthyroidism itself appears to have an adverse effect on generalizedsuppressor T-cell function, and this may be a self-perpetuated orpotentiating factor in Graves' disease. Significantly, there is no knowncure for Grave's disease, treatment being designed merely to reduce thethyroid's ability to produce thyroid hormones.

Causes of hyperthyroidism besides Graves' disease, include toxicmultinodular goiter, toxic adenoma, subacute viral thyroiditis,postpartum thyroiditis, thyroid, gonadal and pituitary tumors and excesspituitary TSH.

The normal thyroid gland weighs about fifteen grams. It is convexanteriorly and concave posteriorly as a result of its relation to theanterolateral portions of the trachea and larynx, to which it is firmlyfixed by fibrous tissue. The two lateral lobes of the thyroid extendalong the sides of the larynx, reaching the level of the middle of thethyroid cartilage. Each thyroid lobe resides in a bed between thetrachea and larynx medially and the carotid sheath andstemocleidomastoid muscles laterally.

The thyroid is composed of an aggregation of spherical or ovate cystlikefollicles of variable size. The interfollicular areas are occupied by ahighly vascularized network which includes parafollicular cells (Ccells) which are responsible for the secretion of calcitonin.Parathyroid hormone (PTH, made by the parathyroid glands), calcitonin(made by the C cells of the thyroid) and dihydroxycholecalciferol(metabolized from vitamin D in the kidney) are the principal hormonesconcerned with the metabolism of ions such as calcium, phosphate,pyrophosphate, citrate and magnesium, and with the regulation of themetabolism of bone and its organic constituents. In humans, it isbelieved that calcitonin acts, in a manner antagonistic to PTH, to lowerplasma calcium.

The thyroid gland is enveloped by a thickened fibrous capsule; The deepcervical fascia divides into an anterior and a posterior sheath,creating a loosely applied false capsule for the thyroid. Anterior tothe thyroid lobes are the strap muscles. Situated on the posteriorsurface of the lateral lobes of the thyroid gland are the parathyroidglands and the recurrent laryngeal nerves; the latter usually lie in adeft between the tea and the esophagus. The lateral lobes of the thyroidare joined by the isthmus that crosses the trachea. A pyramidal lobe isoften present. The pyramidal lobe is a long, narrow projection ofthyroid tissue extending upward from the isthmus lying on the surface ofthe thyroid cartilage. It represents a vestige of the embryonicthyroglossal duct.

Thyroid Vascular Supply

The thyroid has an abundant blood supply. Its four major arteries arethe paired superior thyroid art ries, which arise from the externalcarotid arteries and descend several centimeters in the neck to reachthe upper poles of each thyroid lobe, where they branch, and the pairedinferior thyroid arteries, each of which arises from the thyrocervicaltrunk of the subclavian artery, runs medially behind the carotid arteryand enters the lower or midpart of the thyroid lobe from behind. A fifthartery, the thyroidea ima, is sometimes present; it arises from the archof the aorta and enters the thyroid in the midline.

A venous plexus forms under the thyroid capsule. Each lobe is drained bythe superior thyroid vein at the upper pole and the middle thyroid veinat the middle part of the lobe, both of which enter the internal jugularvein. Arising from each lower pole are the inferior thyroid veins, whichdrain directly into the innominate vein.

Thyroid Innervation

Significantly, the thyroid gland receives innervation from both thesympathetic and parasympathetic divisions of the autonomic nervoussystem. The sympathetic fibers arise from the cervical ganglia and enterwith blood vessels, whereas the parasympathetic fibers are derived fromthe vagus and reach the gland via branches of the laryngeal nerves. Thethyroid gland's relation to the recurrent laryngeal nerves and to theexternal branch of the superior laryngeal nerves is of major surgicalsignificance, since damage to these nerves can lead to a disability ofphonation.

Sympathetic innervation of the thyroid cells has been reported to exerta stimulatory effect upon thyroid hormone release through adrenergicreceptors for norepinephrine on follicle cells. Endocrinology1979;105:7-9. Significantly, the human thyroid is also innervated bycholinergic, parasympathetic fibers. Cell Tiss Res 1978;195:367-370. Seealso Biol Signals 1994;3:?15-25. And other mammalian species are knownto also have cholinergicly innervated thyroid cells. See e.g. Z MikroskAnat Forsch Leipzig 1986;100:1,S, 34-38 (pig thyroid is cholinergiclyinnervated); Neuroendocrinology 1991;53:69-74 (rat thyroid ischolinergicly innervated); Endocrinology 1984;114:1266-1271 (dog thyroidis cholinergicly innervated);

It has been reported that stimulation of the vagal nerve increases boththyroid blood flow and thyroid hormone secretion (Cell Tiss Res1978;195:367-370), but this is apparently due to the extensive,generalized effect of vagal stimulation which can trigger a number ofreflexes ascribed to the whole vagus territory. It is thereforeinappropriate to conclude from this observation the vagal stimulation toacts directly upon the thyroid to increase thyroid hormone release.

Significantly, the consensus is that cholinergic, parasympatheticinfluence upon thyroid hormone secretion by thyroid follicle cells, andpresumably also of the intimately associated C cells, in inhibitory.Endocrinology 1979;105:7-9; Endocrinology 1984;114:1266-1271; Peptides1985;6:585-589; Peptides 1987;8:893-897, and; Brazilian J Med Biol Res1994;27:573-599. The direct cholinergic influence upon the thyroidappears to be mediated by muscarinic acetylcholine receptors of thyroidfollicle cells since the cholinergic inhibition is blocked by atropine.Endocrinology 1979;105:7. The proximity of the non-follicular,calcitonin secreting cells of the thyroid to the thyroid hormonesecreting follicle cells has led to the conclusion that parasympatheticinfluence over the C cells is also inhibitory.

Current Therapy

Therapy for thyroid disorders includes systemically administered drugs,radiotherapy and surgical resection. Unfortunately, all three of thesecurrent therapeutic procedures to treat thyroid disorders, includingGraves' disease, have significant drawbacks and deficiencies.

Drug therapy for hyperthyroidism includes use of the antithyroid drugspropylthiouracil (PTU) and methimazole (Tapazole), both of which inhibitthe organic binding of iodide. In addition, propylthiouracil inhibitsthe peripheral conversion of T₄ to T₃. Notably, the half-life ofpropylthiouracli is only about 1.5 hours while that of methimazole isonly about 6 hours. The initial dose of propylthiouracil is 200 to 300mg, up to 1200 mg daily, every 8 to 12 hours or every 4 to 6 hours whenlarge doses are required. The usual regimen of methimazole is 20 to 40mg daily in one to three divided doses. Such drug therapy is usuallyprescribed for 12 to 18 months.

As Is well known, intravenous, oral or other systemic routes for drugadministration can cause many undesirable side effects, includingnausea, diarrhea and drug resistance. Additionally, the patient mustremember to take the medication. While the antithyroid drugs fortreating hyperthyroidism, such as carbimazole, methimazole andpropylthiouracil, suppress the ability of the thyroid to make hormonesand can render the patient euthyroid, they can also can cause nausea,indigestion, skin rashes, joint pain, fever, and lymphatic glandswelling. Additional side effects include neutropenia andagranulocytosis. Beta adrenergic blocking drugs, such as propranolol(taken orally several times a day), have been used to treat secondarysymptoms of hyperthyroidism, such as excessive sweating, tachycardia,hand tremors and anxiety. Iodine has also been used to treathyperthyroidism due to its a suppressive effect upon the release ofthyroid hormones. Unfortunately, this effect lasts for only about 3 or 4weeks.

Radioactive iodine therapy using ¹³¹I therapy is designed to administera sufficient radiation dose to partially destroy the thyroid parenchyma.Biologic effects of ¹³¹I include pyknosis and necrosis of the follicularcells and, later, vascular and stromal fibrosis. The ¹³¹I dose, inmicrocuries (μCi), to deliver is calculated using the formula: weight ofthyroid gland (gm) X dose (μCi/gm)/uptake (%). The weight of the thyroidgland is estimated by palpation and , the 24 hour iodine uptake ismeasured using a tracer dose of ¹²³I The dose of ¹³¹I that is used fortreatment of Graves' disease ranges from 70 to 215 μCi/gm. Higher dosesare associated with less relapse but can also be associated with ahigher incidence of hypothyroidism during the first few years followingtreatment.

Radiation therapy, such as by use of radioactive iodine (¹³¹I) whichconcentrates in the thyroid, can by irradiation destroy healthy tissueand cause toxicity reactions, and radiation therapy can have potentiallyharmful effect. Notably, iodine therapy is not used beyond the firsttrimester of pregnancy or in children under 15. Additionally, it cantake 2 or 3 months before an effect of radio-iodine treatment becomesapparent. Furthermore, the destruction of thyroid cells by radiationtherapy can result in hyperthyroidism.

Significantly, about 80 percent of Graves' disease patients treated with¹³¹I subsequently become hypothyroid. Additionally, there is evidencethat the treatment of thyroid overactivity in Graves' disease withradio-iodine may aggravate the various ophthalmopathic conditions morethan does treatment with an antithyroid drug. Bayliss et al., supr pages69-70.

The usual surgical treatment of Graves disease consists of subtotalthyroidectomy leaving 3 to 5 grams of residual thyroid tissue attachedto an intact inferior thyroid artery. The choice of therapy may beinfluenced by cost, age, the size of the goiter, the degree ofthyrotoxicosis, pregnancy status, patient preferences, and response toinitial treatment Surgery, because of the potential complications andthe cosmetic effect has only a minimal role in the treatment of Graves'disease and is recommended only in patients for whom other therapies arecontraindicated or refused.

The surgical option, that is thyroidectomy, has been performed astherapy for thyrotoxicosis, to remove benign and malignant tumors, toalleviate pressure symptoms or respiratory obstruction attributable tothe thyroid, and occasionally, to remove an unsightly goiter. Videoassisted thyroidectomy has been used to minimize the length of incisionsin the neck or to hide the incisions by placing them below the clavicleor far lateral in the neck. The recurrent laryngeal nerve andparathyroid glands can be seen as the thyroid lobectomy is performed.While surgical removal of disfunctional thyroid tissue can be aneffectivve therapy, it is irreversible and depends to a large extentupon the skill of the surgeon. Additionally, some thyroids tumors andcancers are inoperable due to proximity or attachment to vitalstructures. Furthermore, although the mortality rate accompanyingthyroidectomy is very low (reported as 0.19 percent), the morbidity rateis about 13 percent when all complications, including the most minortypes are considered.

Complications ensuing from surgery have included hypothyroidism, thyroidstorm, wound infection, wound hemorrhage with hematoma formation,recurrent laryngeal nerve injury, hypoparathyroldism and tracheomalacia.Significantly, following total or near-total thyroidectomy patients musttake thyroid hormone replacement for the remainder of their lives orsuffer severe symptoms and signs of myxedema, including tiredness,weakness, depression, psychosis, mental retardation, coma and death.

Notably, about 20% of hyperthyroid patients who have had a surgicalthyroidectomy become hypothyroid within one year. Additionally, damageto the recurrent laryngeal nerves during surgery can cause permanenthoarseness of the voice. Furthermore, intraoperative damage to theparathyroids can cause blood calcium levels to fall with resultingtetany.

Thyroid underactivity can be due to a dietary lack of iodine, the lackof which prevents thyroid cell synthesis of the thyroid hormones. In theWestern world, Hashimoto's disease is the most common cause ofhypothyroidism. Hypothyroidism can also result from radio-iodinetreatment or surgery to correct thyroid overactivity, as well as to apituitary disorder. The treatment of choice for hypothyroidism isreplacement therapy with thyroxine. Treatment of hypothyroidism bythyroid hormone replacement requires long term, daily dosing withexpensive medication from which undesirable side effects can occur.

Ophthalmopathy

Hyperthyroidism can result in an overactivity of the sympathetic nervoussystem with resulting eyelid retraction. Other ophthalmic disorders canbe the result of autoimmune hyperthyroidism (i.e. Graves' disease andHashimoto's thyroiditis) when the autoantibodies also affect ocularmuscles. Thus, proptosis is or exophthalmos (eyes pushed forwards),impaired eye fluid drainage which can cause increased fluid pressure andblindness, ophthalmopegia (impaired eye muscle control), diplopia(double vision), and blindness. Therapeutic approaches have includedbeta blockers to reduce lid retraction, surgery to lower the eyelids andto correct the diplopia, corticosteroids (such as prednisone andmethylprednisolone) to reduce eye protrusion by suppressing the ocularautoimmune reaction, and X-raying the orbits and surgery to increase thesize of the orbits.

Significantly, and as indicated, treatment of hyperthyroidism with ¹³¹Ican exacerbate ophthalmopathy more than does treatment with anantithyroid drug. Hence, an effective drug treatment can be preferred touse of radio-iodine or surgery with all its potential complications andrequired skill level.

Thus, each of the drug, radioactive iodine and surgical therapies fortreating thyroid disorders has significant attendant risks,complications, drawbacks and deficiencies. Presently available drugshave only an antithyroid, as opposed to a prothyroid effect, and areadministered systemically. Clearly, there is a considerable need for aneffective antithyroid drug to treat hyperthyroidism and for a suitablealternative to thyroid hormone replacement to treat hypothyroidism.

Botulinum Toxin

The anaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinIntoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of a commercially available botulinum toxintype A (purified neurotoxin complex)¹ is a LD₅₀ In mice (i.e. 1 unit).Interestingly, on a molar basis, botulinum toxin type A is about 1.8billion times more lethal than diphtheria, about 600 million times morelethal than sodium cyanide, about 30 million times more lethal thancobra toxin and about 12 million times more lethal than cholera. Singh,Critical Aspect of Bacterial Protein Toxins, pages 63-84 (chapter 4) ofNatural Toxins II, edited by B. R. Singh et al., Plenum Press, New York(1996) where the stated LD₅₀ of botulinum toxin type A of 0.3 ng equals1 U is corrected for the fact that about 0.05 ng of BOTOX® equals 1unit). One unit (U) of botulinum toxin is defined as the LD₅₀ uponintraperitoneal injection into female Swiss Webster mice weighing 18 to20 grams each.

¹Available from Allergan, Inc. of Irvine, Calif. under the tradenameBOTOX® in 100 unit vials. One unit of BOTOX® contains about 50 picogramsof botulinum toxin A complex.

Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G each of which is distinguished by neutralizationwith type-specific antibodies. The different serotypes of botulinumtoxin vary in the animal species that they affect and in the severityand duration of the paralysis they evoke. For example, it has beendetermined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Botulinum toxinapparently binds high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A has been approved by the U.S. Food andDrug Administration for the treatment of blepharospasm, strabismus andhemifacial spasm. Nontype A botulinum toxin serotypes apparently have alower potency and/or a shorter duration of activity as compared tobotulinum toxin type A. Clinical effects of peripheral intramuscularbotulinum toxin type A are usually seen within one week of injection.The typical duration of symptomatic relief from a single intramuscularinjection of botulinum toxin type A averages about three months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites, For example, botulinum typesA and E both cleave the 25 kiloDalton (kD) synaptosomal associatedprotein (SNAP-25), but they target different amino acid sequences withinthis protein. Botulinum toxin types B, D. F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C, has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.Significantly, it is known that the cytosol of pancreatic islet B cellscontains at least SNAP-25 (Biochem J 1;339 (pt 1): 159-65 (April 1999)),and synaptobrevin (Mov Disord 1995 May; 10(3): 376).

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 500 kD complex. Botulinum toxin type D is produced asboth 300 kD and 500 kD complexes. Finally, botulinum toxin types E and Fare produced as only approximately 300 kD complexes. The complexes (i.e.molecular weight greater than about 150 kD) are believed to contain anon-toxin hemaglutinin protein and a non-toxin and non-toxicnonhemaglutinin protein. These two non-toxin proteins (which along withthe botulinum toxin molecule comprise the relevant neurotoxin complex)may act to provide stability against denaturation to the botulinum toxinmolecule and protection against digestive acids when toxin is ingested.Additionally, it is possible that the larger (greater than about 150 kDmolecular weight) botulinum toxin complexes may result in a slower rateof diffusion of the botulinum toxin away from a site of intramuscularinjection of a botulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, a has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains is that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of Inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≧3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Schantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Schantz E. J. et al.Properties and use of Botulinum toxin and Other Microbial Neurotoxins inMedicine, Microbiol Rev. 56: 80-99 (1992). Generally, the botulinumtoxin type A complex can be isolated and purified from an anaerobicfermentation by cultivating Clostridium botulinum type A in a suitablemedium. The known process can also be used, upon separation out of thenon-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin typo F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Already prepared and purified botulinum toxins and toxin complexes canbe obtained from List Biological Laboratories, Inc., Campbell, Calif.;the Centre for Applied Microbiology and Research, Porton Down, U. K.;Wako (Osaka, Japan), as well as from Sigma Chemicals of St. Louis, Mo.

Pure botulinum toxin is so labile that i is generally not used toprepare a pharmaceutical composition. Furthermore, the botulinum toxincomplexes, such the toxin type A complex are also extremely susceptibleto denaturation due to surface denaturation, heat, and alkalineconditions. Inactivated toxin forms toxoid proteins which may beimmunogenic. The resulting antibodies can rend r a patient refractory totoxin injection.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin must be formulated with astabilizing agent, such as albumin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. The botulinum toxin type A is made from a culture ofthe Hall strain of Clostridium botulinum grown In a medium containingN-Z amine and yeast extract. The botulinum toxin type A complex ispurified from the culture solution by a series of acid precipitations toa crystalline complex consisting of the active high molecular weighttoxin protein and an associated hemagglutinin protein. The crystallinecomplex is re-dissolved in a solution containing saline and albumin andsterile filtered (0.2 microns) prior to vacuum-drying. BOTOX® can bereconstituted with sterile, nonpreserved saline prior to intramuscularinjection. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A purified neurotoxin complex, 0.5milligrams of human serum albumin and 0.9 milligrams of sodium chloridein a sterile, vacuum-dried form without a preservative.

BOTOX® can be reconstituted with 0.9% Sodium Chloride Injection. SinceBOTOX® can be denatured by bubbling or similar violent agitation, thediluent is gently injected into the vial. BOTOX® should be administeredwithin four hours after reconstitution. During this time period,reconstituted BOTOX® is stored in a refrigerator (2° to 8° C.).Reconstituted BOTOX® is clear, colorless and free of particulate matter.The vacuum-dried product is stored in a freezer at or below −5° C.BOTOX® is administered within four hours after the vial is removed fromthe freezer and reconstituted. During these four hours, reconstitutedBOTOX® can be stored in a refrigerator (2° to 8° C.). ReconstitutedBOTOX® is clear, a colorless and free of particulate matter.

It has been reported that botulinum toxin type A has been used inclinical a settings as follows:

(1) about 75-125 units of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectails muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

(a) flexor digitorum profundus: 7.5 U to 30 U

(b) flexor digitorum sublimus: 7.5 U to 30 U

(c) flexor carpi ulnaris: 10 U to 40 U

(d) flexor carpi radians: 15 U to 60 U

(e) biceps brachii: 50 U to 200 U. Each of the five indicated muscleshas been injected at the same treatment session, so that the patientreceives from 90 U to 360 U of upper limb flexor muscle BOTOX® byintramuscular injection at each treatment session.

(7) to treat migraine, pericranial injected (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

The success of botulinum toxin tpye A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Astudy of two commercially available botulinum type A preparations(BOTOX® and Dysport®) and preparations of botulinum toxins type B and F(both obtained from Wako Chemicals, Japan) has been carried out todetermine local muscle weakening efficacy, safety and antigenicpotential. Botulinum toxin preparations were injected into the head ofthe right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscleweakness was assessed using the mouse digit abduction scoring assay(DAS). ED₅₀ values were calculated from dose response curves. Additionalmice were given intramuscular injections to determine LD₅₀ doses. Thetherapeutic index was calculated as LD₅₀/ED₅₀. Separate groups of micereceived hind limb injections of BOTOX® (5.0 to 10.0 units/kg) orbotulinum toxin type B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antgenic potential was assessed by monthlyintramuscular injections in rabbits (1.5 or 6.5 ng/kg for botulinumtoxin type B or 0.15 ng/kg for BOTOX®). Peak muscle weakness andduration were dose related for all serotypes. DAS ED₅₀ values (units/kg)were as follows: BOTOX®: 6.7, Dysport®: 24.7, botulinum toxin type B:27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX® had a longer durationof action than botulinum toxin type B or botulinum toxin type F.Therapeutic index values were as follows: BOTOX®: 10.5, Dysport®: 6.3,botulinum toxin type B: 3.2. Water consumption was greater in miceinjected with botulinum toxin type 8 than with BOTOX®, althoughbotulinum toxin type B was less effective at weakening muscles. Afterfour months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of4 (where treated with 6.5 ng/kg) rabbits developed antibodies againstbotulinum toxin type B. In a separate study, 0 of 9 BOTOX® treatedrabbits demonstrated antibodies against botulinum toxin type A. DASresults indicate relative peak potencies of botulinum toxin type A beingequal to botulinum toxin type F, and botulinum toxin type F beinggreater than botulinum toxin type B. With regard to duration of effect,botulinum toxin type A was greater than botulinum toxin type B, andbotulinum toxin type B duration of effect was greater than botulinumtoxin type F. As shown by the therapeutic index values, the twocommercial preparations of botulinum toxin type A (BOTOX®and Dysport®)are different. The increased water consumption behavior observedfollowing hind limb injection of botulinum toxin type B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to botulinum toxin type A, it is necessary toincrease doses of the other serotypes examined. Increased dosage cancomprise safety. Furthermore, in rabbits, type B was more antigenic thanwas BOTOX®, possibly because of the higher protein load injected toachieve an effective dose of botulinum toxin type B. Eur J Neurol 1999November;6(Suppl 4):S3-S10.

Acetylcholine

Typically only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine Is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition ofheart rate by the vagal nerve.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic, neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic neurons of the parasympathetic nervoussystem, as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the synapses between the preganglionic and postganglionicneurons of both the sympathetic and parasympathetic. The nicotinicreceptors are also present in many membranes of skeletal muscle fibersat the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and thyroid hormone, respectively, from large dense-corevesicles. The PC12 cell line is a clone of rat pheochromocytoma cellsextensively used as a tissue culture model for studies ofsympathoadrenal development. Botulinum toxin inhibits the release ofboth types of compounds from both types of cells in vitro, permeabilized(as by electroporation) or by direct injection of the toxin into thedenervated cell. Botulinum toxin is also known to block release of theneurotransmitter glutamate from cortical synaptosomes cell cultures.

What is needed therefore is an effective, long lasting, non-surgicalresection, non-radiotherapy, non-systemic drug administration,therapeutic drug and method for treating thyroid disorders.

SUMMARY

The present invention meets this need and provides an effective,non-surgical resection, relatively long tern, non-radiotherapy,non-systemic drug administration, therapeutic method for treatingthyroid disorders. The drug within the scope of this invention fortreating thyroid disorders is a neurotoxin. Significantly, the sameneurotoxin can be used to treat hypothyroidism, hyperthyroidism,hypocalcemia and hypercalcemia depending upon factors such as the siteof local administration of the neurotoxin and the amount of neurotoxinto be administered.

As used herein “local administration” means direct injection of aneurotoxin into the thyroid or into a sympathetic ganglion whichinnervates a thyroid cell (such as a thyroid follicle cell or thyroid Ccell). Systemic routes of is administration, such as oral andintravenous routes of administration, are excluded from the scope of“local administration” of a neurotoxin.

As used herein, Thyroid hormones means thyroxine (T₄), while “thyroidhormones” means triiodothyronine (T₃) and thyroxine (T₄)

A method for treating a thyroid disorder according to the presentinvention can be carried out by administration of a therapeuticallyeffective amount of a neurotoxin to a patient, thereby treating thethyroid disorder. The neurotoxin can administered to the thyroid of thepatient when the thyroid disorder to be treated is hypothyroidism.Alternately, the neurotoxin can be administered to a sympatheticganglion which innervates the thyroid when the thyroid disorder to betreated is hyperthyroidism.

A detailed method for treating a thyroid disorder according to thepresent invention can comprise the step of administration of atherapeutically effective amount of a botulinum toxin to a patient Thus,a method for treating hypothyroidism according to the present inventioncan comprise the step of local administration to the thyroid of atherapeutically effective amount of a botulinum toxin, therebyincreasing a deficient thyroid hormone secretion from the thyroid celland effectively treating the hypothyroidism. Furthermore, a methodwithin the scope of the present invention for treating hyperthyroidism,can comprise the step of local administration to a sympathetic ganglionwhich innervates a thyroid cell of a therapeutically effective amount ofa botulinum toxin, thereby reducing an excessive thyroid hormonesecretion from the thyroid cell and hence effectively treating thehyperthyroidism.

The neurotoxin can be administered in an amount of between about 10⁻³U/kg and about 35 U/kg. 35 U/kg is an upper limit because it approachesa lethal dose of certain neurotoxins, such as botulinum toxin type A.Other botulinum toxins, such as botulinum toxin type B, can be safelyadministered at several orders of magnitude higher dosage. Preferably,the neurotoxin is administered in an amount of between about 10⁻² U/kgand about 25 U/kg. More preferably, the neurotoxin is administered in anamount of between about 10⁻¹ U/kg and about 15 U/kg. Most preferably,the neurotoxin is administered in an amount of between about 1 U/kg andabout 10 U/kg. In many instances, an administration of from about 1units to about 500 units of a neurotoxin, such as a botulinum toxin typeA, provides effective and long lasting therapeutic relief. Morepreferably, from about 5 units to about 300 units of a neurotoxin, suchas a botulinum toxin type A, can be used and most preferably, from about10 units to about 200 units of a neurotoxin, such as a botulinum toxintype A, can be locally administered into a target tissue such as thethyroid or a sympathetic ganglion with efficacious results. In aparticularly preferred embodiment of the present invention from about 10units to about 100 units of a botulinum toxin, such as botulinum toxintype A, can be locally administered into a target tissue such as thethyroid or a sympathetic ganglion with therapeutically effectiveresults.

The neurotoxin can be made by a Clostridial bacterium, such as by aClostridium botulinum, Clostridium butyricum, Clostridium beratti orClostridium tetani bacterium. Additionally, the neurotoxin can be amodified neurotoxin, that is a neurotoxin that has at least one of itsamino acids deleted, modified or replaced, as compared to the native orwild type neurotoxin. Furthermore, the neurotoxin can be a recombinantproduced neurotoxin or a derivative or fragment thereof.

The neurotoxin can be a botulinum toxin, such as one of the botulinumtoxin serotypes A, B, C₁, D, E, F or G. Preferably, the neurotoxin isbotulinum toxin type A and the neurotoxin is locally administered bydirect injection of the neurotoxin into the thyroid or into asympathetic ganglion which innervates the a thyroid.

A detailed embodiment of a method within the scope of the presentinvention for treating a thyroid disorder can comprise the step ofinjecting a therapeutically effective amount of a botulinum toxin into athyroid of a human patient, thereby increasing a thyroid hormonesecretion from a thyroid cell and treating a thyroid disorder.

Another detailed embodiment of a method within the scope of the presentinvention for treating a thyroid disorder of a human patient cancomprise the step of local administration to a cholinergic influencedthyroid cell of a human patient of a therapeutically effective amount ofbotulinum toxin type A, thereby increasing a cholinergic influenceddeficient thyroid hormone secretion from the thyroid cell and treatingthe thyroid disorder.

Another method within the scope of the present invention is a method fortreating a thyroid disorder by administration of a neurotoxin to asympathetic nervous system of a patient. In this method the neurotoxinis locally administered to a sympathetic ganglion which innervates athyroid cell and the thyroid disorder is hyperthyroidism.

A detailed embodiment of a method within the scope of the presentinvention for treating a thyroid disorder of a human patient cancomprise the step of in vivo, local administration to a sympatheticganglion, which innervates a thyroid cell of a patient, of atherapeutically effective amount of a botulinum toxin, therebydecreasing an excessive thyroid hormone secretion from a thyroid celland treating hyperthyroidism.

A detailed embodiment of the present invention is a method for treatinga thyroid disorder by Injecting a therapeutically effective amount of abotulinum toxin into a thyroid of a human patient, thereby increasing asecretion of a thyroid hormone from a thyroid cell and treating thethyroid disorder. Preferably, the secretion treated is a cholinergicinfluenced secretion and the botulinum toxin a used is botulinum toxintype A, although the botulinum toxin can selected from the groupconsisting of botulinum toxin types A, B, C (i.e. C₁), D, E, F and G.

My invention also Includes within its scope, a method for treatinghypercalcemia, the method comprising the step of local administration toa thyroid C cell of a therapeutically effective amount of a botulinumtoxin, thereby increasing a deficient calcitonin secretion from athyroid C cell and treating hypercalcemia. Additionally, my inventionalso includes within its scope a method for treating hypocalcemia, themethod comprising the step of local administration to a sympatheticganglion which innervates a thyroid C cell of a therapeuticallyeffective amount of a botulinum toxin, thereby increasing a deficientcalcitonin secretion from the thyroid C cell and treating hypocalcemia.

DESCRIPTION

The present invention is based upon the discovery that a thyroiddisorder can be treated by in vivo administration of a neurotoxin to apatient. Thus administration of a neurotoxin to the thyroid of a patientcan remove an inhibitory cholinergic effect upon thyroid hormonesecretion, thereby providing an effective treatment for hypothyroidism.Additionally, administration of a neurotoxin to a sympathetic ganglionwhich innervates the thyroid can remove a stimulatory adrenergic effectupon thyroid hormone secretion, thereby providing an effective treatmentfor hyperthyroidism.

Thus, thyroid disorders can be treated, according to the presentinvention, by the alternative therapies of (a) local administration of aneurotoxin to the thyroid, or; (b) local administration of a neurotoxinto a sympathetic ganglion of a patient, thereby resulting in,respectively, an increase of a secretion from a thyroid cell, or adecrease in a secretion from a thyroid cell.

I have discovered that a particular neurotoxin, botulinum toxin, can beused with dramatic ameliorative effect to treat a thyroid disorder,thereby significantly superseding thereby current therapeutic regimens,such as oral thyroid hormone (to treat hypothyroidism) or radioactiveiodine (to treat hyperthyroidism). Significantly, a single localadministration of a neurotoxin, such as a botulinum toxin to thethyroid, according to the present invention, can increase thyroidhormone secretion and thereby treat symptoms of hypothyroidism. I havealso discovered that a single local administration of a neurotoxin, suchas a botulinum toxin to a sympathetic ganglion which innervates thethyroid gland, according to the present invention, can reduce thyroidhormone secretion and thereby treat symptoms of hyperthyroidism. Ineither case, the symptoms of the thyroid disorder can be alleviated forat least about from 2 months to about 6 months per neurotoxinadministration. Notably, it has been reported that glandular tissuetreated by a botulinum toxin can show a reduced secretory activity foras long as 27 months post injection of the toxin. Laryngoscope 1999;109:1344-1346, Laryngoscope 1998; 108:381-384.

The hypothyroidism treatable by the present invention is hypothyroidismwhich has as a causative factor the inhibitory activity upon thyroidhormone secretion of parasympathetic innervation of the thyroid. Thus,treatment of hypothyroidism which results directly and solely from, forexample, a dietary iodine deficiency or from the action of antithyroidantibodies, is outside the scope of the present invention. Similarly,the hyperthyroidism treatable by the present invention ishyperthyroidism which has as a causative factor the stimulatory activityupon thyroid hormone secretion of sympathetic innervation of thethyroid. Thus, treatment of hyperthyroidism which results directly andsolely from, for example, the effect of thyroid stimulating antibodiesupon the thyroid is outside the scope of the present invention.

Notably, hypothyroidism resulting from a combination of factors,including Tar, inhibitory parasympathetic activity, is treatable by amethod within the scope of the present invention. Similarly,hyperthyroidism resulting from a combination of factors, includingstimulatory sympathetic activity, is treatable by a method within thescope of the present invention.

Local Administration of a Neurotoxin to the Thyroid

A preferred embodiment of the present invention is to inject the thyroidof a patient with from 1 to 500 units, more preferably from 10 to 200units, and most preferably from 20 to 100 units of a neurotoxin (such asa botulinum toxin type A), to thereby cause a reduction of thyroidfollicle hormone secretion. The present invention also includes withinits scope treatment of a thyroid disorder due to hyperplasic, hypertonicor hypertrophic thyroid follicle cells. A thyroid disorder can beeffectively treated by local administration of a n urotoxin, such as forexample 10 to 500 units of botulinum toxin type A, to cholinergic,postganglionic, parasympathetic neurons which innervate thedysfunctional, thyroid cells. Without wishing to be bound by theory, thebotulinum toxin is believed to act by inhibiting release ofacetylcholine neurotransmitter from cholinergic, postganglionicparasympathetic fibers which innervate thyroid follicle cells.

A neurotoxin, such as a botulinum toxin, can be locally administered invivo to the thyroid to thereby remove an inhibitory effect upon asecretory activity of a thyroid follicle cell. The thyroid follicle cellis cholinergically innervated or susceptible to high toxin dosing suchthat the proteolytic light chain of the toxin is internalized by acholinergic neuron which influences a secretory activity of the thyroidcell.

Thus, cholinergically innervated thyroid cells can be treated by localadministration of a neurotoxin, such as a botulinum toxin. By localadministration it is meant that the neurotoxin is administered directlyto or to the immediate vicinity of the thyroid tissue to be treated.

The specific dosage appropriate for administration is readily determinedby one of ordinary skill in the art according to the factor discussedabove. The dosage can also depend upon the size of the thyroid tissuemass to be treated or denervated, and the commercial preparation of thetoxin. Additionally, the estimates for appropriate dosages in humans canbe extrapolated from determinations of the amounts of botulinum requiredfor effective denervation of other tissues. Thus, the amount ofbotulinum A to be injected is proportional to the mass and level ofactivity of the thyroid tissue to be treated. Generally, between about0.01 and 35 units per kg of patient weight of a botulinum toxin, such asbotulinum toxin type A, can be administered to effectively accomplish atoxin induced thyroid tissue secretion up regulation upon administrationof the neurotoxin into the thyroid. Less than about 0.01 U/kg of abotulinum toxin does not have a significant therapeutic effect upon thesecretory activity of a thyroid cell, while more than about 35 U/kg of abotulinum toxin approaches a toxic dose the neurotoxin. Carefulplacement of the injection needle and a low volume of neurotoxin usedprevents significant amounts of botulinum toxin from appearingsystemically. A more preferred dose range is from about 0.01 U/kg toabout 25 U/kg of a botulinum toxin, such as that formulated as BOTOX®.The actual amount of U/kg of a botulinum toxin to be administereddepends upon factors such as the extent (mass) and level of activity ofthe thyroid tissue to be treated and the administration route chosen.Botulinum toxin type A is a preferred botulinum toxin serotype for usein the methods of the present invention.

It has been reported that the neuronal selectivity of clostridialneurotoxins is a result of a very selective binding and cell entrymechanism. Although a site of action of botulinum toxin is theneuromuscular junction, where the toxin binds rapidly and prevents therelease of acetylcholine from cholinergic neurons, it is known thatclostridial neurotoxins are able to enter certain neurosecretory cells(for example PC12 cells) via a low affinity receptor if highconcentrations of the neurotoxin are incubated with the cells forprolonged periods. This process appears to use a pathway via a receptorwhich is distinct from the highly specific and high affinity receptorpresent at the neuromuscular junction. Additionally, it has beenreported that certain clostridial toxins have effects on phagocytecells, such as macrophages, where entry into the cell is presumed to bevia the specific phagocytic activity of these cells. Furthermore,incubation of certain adipocytes (i.e. fat cells) with botulinum toxintype A has been reported to inhibit glucose uptake by the adipocytes.The mechanism of the glucose uptake inhibition is apparently due totoxin inhibition of plasma membrane fusion or docking of cytosolic,recyclable membrane vesicles (RMVs), the RMVs containing glucosetransporter proteins. PCT publication WO 94/21300.

Thus, while it is known that the botulinum toxins have a bindingaffinity for cholinergic, pre-synaptic, peripheral motor neurons, it hasbeen reported that botulinum toxins can also bind to and translocateinto a variety of non-neuronal secretory cells, where the toxin thenacts, in the known manner, as an endoprotease upon its respectivesecretory vessel-membrane docking protein. Because of the relativelylower affinity of the botulinum toxins for secretory cells, such asthyroid cells, as compared to the affinity of the botulinum toxin forthe cholinergic neurons which innervate thyroid cells, the botulinumtoxin can be injected into secretory or glandular tissues to provide ahigh local concentration of the toxin, thereby facilitating effect ofthe toxin upon both cholinergic neuron and directly upon thyroidsecretory cell. Thus, the present invention is applicable to thetreatment of thyroid disorders wherein the target thyroid cells havelittle or no cholinergic innervation.

Preferably, a neurotoxin used to practice a method within the scope ofthe present invention is a botulinum toxin, such as one of the serotypeA, B, C, D. E, F or G botulinum toxins. Preferably, the botulinum toxinused is botulinum toxin type A, because of its high potency in humans,ready availability, and known safe and efficacious use for the treatmentof skeletal muscle and smooth muscle disorders when locally administeredby intramuscular injection.

The present invention includes within its scope the use of anyneurotoxin which has a long duration therapeutic effect when locallyapplied to treat a thyroid cell disorder of a patient For example,neurotoxins made by any of the specie of the toxin producing Clostridiumbacteria, such as Clostridium botulinum, Clostridium butyricum, andClostridium beratti can be used or adapted for use in the methods of thepresent invention. Additionally, all of the botulinum serotypes A, B, C,D, E, F and G can be advantageously used in the practice of the presentinvention, although type A Is the most preferred serotype, as explainedabove. Practice of the present invention can provide effective relief ofa thyroid disorder for from 2-27 months or longer in humans.

Botulinum toxin is believed to be able to block the release of anyvesicle mediated exocytosis from any secretory (i.e. neuronal,glandular, secretory, chromaffin) cell type, as long as the light chainof the botulinum toxin is translocated into the intracellular medium.For example, the intracellular protein SNAP-25 is widely distributed inboth neuronal and non-neuronal secretory cells and botulinum toxin typeA is an endopeptidase for which the specific substrate is SNAP-25. Thus,while cholinergic neurons have a high affinity acceptor for thebotulinum and tetanus toxins (and are therefore more sensitive thanother neurons and other cells to the inhibition of vesicle mediatedexocylosis of secretory compounds), as the toxin concentration israised, non-cholinergic sympathetic neurons, chromaffin cells and othercell types can take up a botulinum toxin and show reduced exocytosis.

Hence, by practice of the present disclosed invention, non-cholinergicnerve fibers as well as non or poorly innervated thyroid cells can betreated by use of an appropriately higher concentration of a botulinumtoxin to bring about therapeutic relief from a thyroid disorder.

Local Administration of a Neurotoxin to a Sympathetic Ganglion

Significantly, a method within the scope of the present invention forreducing an excessive thyroid hormone secretion comprises the step oflocal administration of a neurotoxin to the sympathetic nervous system.Sympathetic innervation of the thyroid is know to exist. Thus,sympathetic nerve fibers can stimulate thyroid hormone secretion byacting via adrenergic receptors on thyroid follicles. A method withinthe scope of the present invention can therefore be carried out by localadministration of a neurotoxin to a cholinergic, preganglionicsympathetic neuron. The cholinergic, preganglionic, sympathetic neuronssynapse with adrenergic, postanglionic, sympathetic fibers, and theselater sympathetic neurons have a stipulatory effect upon thyroid hormonesecretion by thyroid cells. Preferably, the sympathetic ganglion towhich a neurotoxin is administered, according to the preset invention,is a cervical ganglion.

Cervical ganglion block according to the present invention can becarried out in the same manner as a celiac plexus block. Thus, theneurolytic celiac plexus block is a known procedure for treatingintractable pain resulting from upper abdominal viscera cancer. RegAnest Pain Med 1998; 23(1):37-48. Thus, it is known to inject the celiacplexus with ethanol or phenol to provide relief from the pain which canresult from pancreatic cancer or from pancreatitis. AJG1999;94(4):872-874. Hence, an antinociceptive injection of the cervicalganglia can be carried out as by either a percutaneous procedure or asan open (intraoperative) injection. The percutaneous (closed) procedurecan be carried out using an anterior approach using a very thin needle(22 Gauge). Cervical ganglion block is preferably carried out withcomputed tomography (CT) (as opposed to fluoroscopic) needle guidance,using a single thin needle.

Furthermore, a method within the scope of the present invention canprovide improved patient function. “Improved patient function” can bedefined as an improvement measured by factors such as a reduced pain,reduced time spent in bed, increased ambulation, healthier attitude,more varied lifestyle and/or healing permitted by normal muscle tone.Improved patient function is synonymous with an improved quality of life(QOL). QOL can be assesses using, for example, the known SF-12 or SF-36health survey scoring procedures. SF-36 assesses a patients physical andmental health in the eight domains of physical functioning, rolelimitations due to physical problems, social functioning, bodily pain,general rental health, role limitations due to emotional problems,vitality, and general health perceptions. Scores obtained can becompared to published values available for various general and patientpopulations.

As set forth above, I have discovered that a surprisingly effective andlong lasting therapeutic effect can be achieved by local administrationof a neurotoxin to the thyroid or to a sympathetic ganglion whichinnervates the thyroid of a human patient In its most preferredembodiment, the present invention is practiced by direct injection intothe thyroid or into the sympathetic ganglion of botulinum toxin type A.It has been reported that at the neuroglandular junction, the chemicaldenervation effect of a botulinum toxin, such as botulinum toxin type A,has a long duration of action, i.e. 27 months vs. 3 months.

The present invention includes within its scope: (a) neurotoxin complexas well as pure neurotoxin obtained or processed by bacterial culturing,toxin extraction, concentration, preservation, freeze drying and/orreconstitution and; (b) modified or recombinant neurotoxin, that isneurotoxin that has had one or more amino acids or amino acid sequencesdeliberately deleted, modified or replaced by known chemical/biochemicalamino acid modification procedures or by use of known hostcell/recombinant vector recombinant technologies, as well as derivativesor fragments of neurotoxins so made, and includes neurotoxins with oneor more attached targeting moieties for a cell surface receptor presenton a thyroid cell.

Botulinum toxins for use according to the present invention can bestored in lyophilized or vacuum dried form in containers under vacuumpressure. Prior to lyophilization the botulinum toxin can be combinedwith pharmaceutically acceptable excipents, stabilizers and/or carriers,such as albumin. The lyophilized or vacuum dried material can bereconstituted with saline or water.

The route of administration and amount of a neurotoxin (such as abotulinum toxin serotype A, B, C, D, E, F or G) administered accordingto the present invention for treating a thyroid disorder can vary widelyaccording to various patient variables including size, weight, age,disease severity, responsiveness to therapy, and solubility anddiffusion characteristics of the neurotoxin toxin chosen. Furthermore,the extent of the thyroid or ganglionic tissue influenced is believed tobe proportional to the volume of neurotoxin injected, while the quantityof the denervation is, for most dose ranges, believed to be proportionalto the concentration of neurotoxin injected.

Methods for determining the appropriate route of administration anddosage are generally determined on a case by case basis by the attendingphysician. Such determinations are routine to one of ordinary skill inthe art (see for example, Harrison's Principles of Internal Medicine(1998), edited by. Anthony Fauci et al., 14^(the) edition, published byMcGraw Hill). For example, to treat a so thyroid disorder, a solution ofbotulinum toxin type A complex can be endoscopically orintraperitoneally injected directly into the tissues of the thyroid,thereby substantially avoiding entry of the toxin into the systemiccirculation.

EXAMPLES

The following examples provide those of ordinary skill in the art withspecific preferred methods within the scope of the present invention forcarrying out the present invention and are not intended to limit thescope of what the inventor regards as his invention. In each of thefollowing examples, the specific amount of a botulinum toxinadministered depends upon a variety of factors to be weighed andconsidered within the discretion of the attending physician and in eachof the examples insignificant amounts of botulinum toxin enter appearsystemically with no significant side effects. Units of botulinum toxininjected per kilogram (U/kg) below are per kg of total patient weight.For example, 3U/kg for a 70 kg patient calls for an injection of 210units of the botulinum toxin.

Example 1 Intraoperative Administration of Neurotoxin

Intraoperative, local administration of a neurotoxin to the thyroid canbe carded out as follows. The procedure can be performed under generalendotracheal anesthesia. The patient's neck can be extended by inflatinga pillow or inserting a thyroid roll beneath the shoulders. Asymmetrical, low, collar incision can then be made in the line of anatural skin crease approximately 1 to 2 cm above the clavicle. Theincision can be carried through the skin, subcutaneous tissue, andplatysma muscle down to the dense cervical fascia that overlies thestrap muscles and anterior jugular veins. The upper flap can then beraised to a level cephalad to the cricoid cartilage. Care is taken toavoid cutting sensory nerves. A small lower flap is also elevated to thelevel of the manubrial notch. Performing dissection of the flaps in theplane between the platysma muscle and the fascia overlying the strapmuscles results in minimal bleeding. The cervical fascia is then incisedvertically in the midline from the thyroid cartilage to the sternalnotch.

Exposure of the superior and lateral aspects of the thyroid gland isachieved by retracting the sternohyoid and sternohyoid muscles laterallyor in very large glands by dividing these muscles. Division of thesemuscles is associated with little or no disability, by is not necessaryunless the gland is markedly enlarged. High transection is preferable,since the ansa cervicalis nerve innervates the muscles from below. Thisprocedure diminishes the amount of muscle that is paralyzed.

Digital or blunt dissection frees the thyroid from the surroundingfascia. As an initial step, the isthmus of the thyroid is usuallyrevealed. Rotation of one lobe of the thyroid can be followed bydissection bluntly. The middle thyroid veins are first encountered andare ligated and divided. This maneuver facilitates exposure of thesuperior and inferior poles of the thyroid lobe. The suspensoryligaments are transected craniad to the isthmus, and the pyramidal lobeand Delphian nodes are mobilized. The cricothyroid space is opened inorder to separate the superior pole from the surrounding tissue. Ifdissection of the superior lobe is to be carried out (i.e. thyroidectomyin conjunction with neurotoxin administration), care is taken to avoidinjury to the superior laryngeal nerve. The internal branch of thenerve, which provides sensory fibers to the epiglottis and larynx, israrely in the operative field. It Is the external branch, which suppliesmotor innervation to the inferior pharyngeal constrictor and thecricothyroid muscle, that must be protected. This purpose is achieved bydissecting the nerve away from the superior pole vessels if it can beidentified, or by separately ligating and dividing the individualbranches of the superior thyroid vessels immediately adjacent to theupper pole of the thyroid lobe rather than cephalad to it.

The lobe can then be retracted mediad to permit identification of theinferior thyroid artery and the recurrent laryngeal nerve. It isessential that meticulous hemostasis be achieved during this part of thedissection. The inferior thyroid artery is isolated, but need not beligated laterally. Rather, when performing a lobectomy, it is preferableto ligate and divide each small arterial branch near the thyroid capsuleat a point after branches to the parathyroid glands have been given off.This technique lessens the incidence of devascularization of theparathyroid gland and plays a role In reducing permanenthypoparathyroidism. If a parathyroid gland is devascularized it can beminced and autotransplanted into the stemocleidomastoid after beingverified by frozen section analysis. The recurrent laryngeal nerve canbe identified along its course by blunt dissection. The fibrous tissuesare gently unroofed from the front of the nerve. The nerve is treatedwith care, for excessive trauma or its division will result in anipsilateral vocal cord paralysis. At the junction of the trachea andlarynx, the recurrent laryngeal nerve is immediately adjacent to thethyroid lobe and is in greatest danger, If It Is not seen. The exposedthyroid lobe can now be directed injected with from 10 to 300 units of abotulinum toxin, such as botulinum toxin type A

During exposure of the posterior surface of the thyroid gland, theparathyroid glands should be identified and preserved, along with theirvascular pedicles. Care should be taken to ensure that the parathyroidglands are not excised or devascularized.

The entire wound is then inspected, and careful hemostasis is obtainedbefore closure. In most instances, a suction catheter is used to drainthe bed of the thyroid lobes, employing a small, soft plastic drain thatis brought out through a stab wound lateral to the incision. Thepostoperative wound appearance with this technique is far superior tothat obtained when no drainage is employed. If the sternohyoid andsternohyoid muscles have been transected, they are reapproximated. Themidline vertical fascial incision is only loosely approximated by oneinterrupted suture, and the drain is positioned superficial to the strapmuscles. There is generally no need to suture the platysma muscle,instead it is preferable to approximate the deep dermis with interrupted4-0 resorbable sutures and the epithelium with 5-0 continuoussubcuticular sutures. Finally, the epithelial surfaces are approximatedwith sterile skin tapes. Within one to seven days, thyroid hormonesecretion is substantially increased due to removal of cholinergicinhibition and this effect persists for from 2 to 6 months.

Example 2 Local Administration of Neurotoxin to the Thyroid

Local administration of a neurotoxin directly to or to the vicinity ofthe thyroid can be accomplished by several methods. For example, bythyroid endoscopy. An endoscope used for thyroid therapy can b modifiedto perm its use for direct injection of a neurotoxin, such as abotulinum toxin directly into thyroid tissue. See for example U.S. Pat.No. 5,674,205. Once appropriately located, a hollow needle tip can beextended from the endoscope into thyroid tissue and through which needlethe neurotoxin can be injected into the thyroid tissue. Additionally,fine needle aspiration for thyroid biopsy purposes is well known and canbe used to inject a neurotoxin, rather than to aspirate thyroid tissue.From 10 to 300 units of a botulinum toxin, such as botulinum toxin typeA can thereby be injected into the thyroid. Within one to seven days,thyroid hormone secretion is substantially increased due to removal ofcholinergic inhibition and this effect persists for from 2 to 6 months.

Example 3 Treatment of Hypothyroidism With Botulinum Toxin Type A

A 43 year old, obese man presents with increasing fatigue over the lasteight months and worsening pedal and calf edema over same time period,which is exacerbated upon standing for very long. Patient also has hadpolydipsia and low urine output over same time period. Notably, TSH is690 (normal is 3-5). A diagnosis of hypothyroidism is made. Betweenabout 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Apreparation (for example between about 10 units and about 300 units ofBOTOX®) is injected directly into the thyroid, using one of thetechniques set forth in Examples 1 or 2 above. Within 1-7 days thesymptoms of the hypothyroidism are alleviated. Thyroid hormone levelsreturn to substantially normal levels. Alleviation of the hypothyroidismpersists for at least about 2 months to about 6 months.

Example 4 Treatment of Hypothyroidism With Botulinum Toxin Type B

A 52 year old female is diagnosed with hypothyroidism. Between about10⁻³ U/kg and about 35 U/kg of a botulinum toxin type B preparation (forexample between about 1000 units and about 20,000 units of a botulinumtype B preparation) is injected directly into the thyroid, using one ofthe techniques set forth in Examples 1 or 2 above. Within 1-7 days thesymptoms of the hypothyroidism are alleviated; Thyroid hormone levelsreturn to substantially normal levels. Alleviation of the thyroiddisorder persists for at least about 2 months to about 6 months.

Example 5 Treatment of Hypothyroidism With Botulinum Toxin Type C

A 58 year old female is diagnosed with hypothyroidism. Between about10⁻³ U/kg and about 35 U/kg of a botulinum toxin type C preparation (forexample between about 10 units and about 10,000 units of a botulinumtype B preparation) is injected directly into the thyroid, using one ofthe techniques set forth in Examples 1 or 2 above. Within 1-7 days thesymptoms of the hypothyroidism are alleviated. Thyroid hormone levelsreturn to substantially normal levels. Alleviation of the thyroiddisorder persists for at least about 2 months to about 6 months.

Example 6 Treatment of Hypothyroidism With Botulinum Toxin Type D

A 56 year old obese female is diagnosed with hypothyroidism. Betweenabout 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Dpreparation (for example between about 10 units and about 10,000 unitsof a botulinum type D preparation) is injected directly into thethyroid, using one of the techniques set forth in Examples 1 or 2 above.Within 1-7 days the symptoms of the hypothyroidism are alleviated.Thyroid hormone levels return to substantially normal levels.Alleviation of the thyroid disorder persists for at least about 2 monthsto about 6 months.

Example 7 Treatment of Hypothyroidism With Botulinum Toxin Type E

A 61 year old female is diagnosed with hypothyroidism. Between about10⁻³ U/kg and about 35 U/kg of a botulinum toxin type E preparation (forexample between about 10 units and about 10,000 units of a botulinumtype E preparation) is injected directly into the thyroid, using one ofthe techniques set forth in Examples 1 or 2 above. Within 1-7 days thesymptoms of the hypothyroidism are alleviated. Thyroid hormone levelsreturn to substantially normal levels. Alleviation of the thyroiddisorder persists for at least about 2 months to about 6 months.

Example 8 Treatment of Hypothyroidism With Botulinum Toxin Type F

A 52 year old male is diagnosed with hypothyroidism. Between about 10⁻³U/kg and about 35 U/kg of a botulinum toxin type F preparation (forexample between about 10 units and about 10,000 units of a botulinumtype F preparation) is injected directly into the thyroid, using one ofthe techniques set forth in Examples 1 or 2 above. Within 1-7 days thesymptoms of the hypothyroidism are alleviated. Thyroid hormone levelsreturn to substantially normal levels. Alleviation of the thyroiddisorder persists for at least about 2 months to about 6 months.

Example 9 Treatment of Hypothyroidism With Botulinum Toxin Type G

A 59 year old female is diagnosed with hypothyroidism. Between about10⁻³ U/kg and about 35 U/kg of a botulinum toxin type G preparation (forexample between about 10 units and about 10,000 units of a botulinumtype G preparation) is injected directly into the thyroid, using one ofthe techniques set forth in Examples 1 or 2 above. Within 1-7 days thesymptoms of the hypothyroidism are alleviated. Thyroid hormone levelsreturn to substantially normal levels. Alleviation of the thyroiddisorder persists for at least about 2 months to about 6 months.

Example 10 Treatment of Hyperthyroidism With Botulinum Toxin Type A

A 27 year old female presents with symptoms and signs ofhyperthyroidism, including thyrotoxicosis and bilateral ophthalmopathy.Thyroid function tests confirmed the diagnosis of hyperthyroidism.Thyroid scintigraphy shows an enlarged gland and indicates thyroidhyperplasia. A trial with atropine reduced the thyroid hormone level.Between about 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Apreparation (for example between about 10 units and about 300 units ofBOTOX®) is injected directly into the cervical ganglia as follows. Apercutaneous procedure is carried out using an anterior approach withthe patient in a supine position using a very thin needle (22 Gauge)with computed tomography needle guidance to reach the cervical ganglia.Within 1-7 days the symptoms of the hyperthyroidism are alleviated.Thyroid hormone levels return to normal (are lowered). Alleviation ofthe a thyroid disorder persists for at least about 2 months to about 6months.

Example 11 Treatment of Hyperthyroidism With Botulinum Toxin Types B-G

A 62 year old female is diagnosed with hyperthyroidism. Between about10⁻³ U/kg and about 35 u/kg of a botulinum toxin type B, C, D, E, F or Gpreparation (for example between about 10 units and about 20,000 unitsof a botulinum toxin type B, C, D, E, F or G preparation) is injecteddirectly into the cervical ganglia as follows. A percutaneous procedureis carried out using an anterior approach with the patient in a supineposition using a very thin needle (22 Gauge) with computed tomographyneedle guidance to reach the cervical ganglia. Within 1-7 days thesymptoms of the hyperthyroidism are alleviated. Thyroid hormone levelsreturn to normal (are lowered). Alleviation of the thyroid disorderpersists for at least about 2 to about 6 months.

Example 12 Treatment of Calcium Metabolism Disorders With BotulinumToxin Types A-G

A 28 year old female is diagnosed with hypercalcemia. Between about 10⁻³U/kg and about 35 U/kg of a botulinum toxin type A, B, C D, E, F or Gpreparation (for example between about 10 units and about 200 units of abotulinum toxin type A preparation) is injected directly into thethyroid using one of the techniques set forth in Examples 1 or 2 above.Within 1-7 days the symptoms of the hypercalcemia are alleviated. Plasmacalcium levels return to substantially normal levels. Alleviation of thehypercalcemia persists for at least about 2 months to about 6 months.

Additionally, to treat hypocalcemia between about 10⁻³ U/kg and about 35U/kg of a botulinum toxin type A, B, C, D, E, F or G preparation (forexample between about 10 units and about 200 units of a botulinum toxintype A preparation) is injected directly into the cervical ganglia asfollows. A percutaneous procedure is carried out using an anteriorapproach with the patient in a supine position using a very thin needle(22 Gauge) with computed tomography needle guidance to reach thecervical ganglia. Within 1-7 days the symptoms of the hypocalcemia arealleviated. Plasma calcium levels return to normal (are increased).Alleviation of the hypocalcemia persists for at least about 2 to about 6months.

Methods according to the invention disclosed herein has many advantages,including the following:

(1) the invention renders unnecessary many surgical procedures foreffective treatment of a thyroid disorder.

(2) systemic drug effects can be avoided by direct local application ofa neurotoxin according to the present invention.

(3) the ameliorative effects of the present invention can persists, onaverage, from about 2 months to about 6 months from a single localadministration of a neurotoxin as set forth herein.

Although the present invention hats been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes local thyroid administration methods wherein two ormore neurotoxins, such as two or more botulinum toxins, are administeredconcurrently or consecutively. For example, botulinum toxin type A canbe administered until a loss of clinical response or neutralizingantibodies develop, followed by administration of botulinum toxin typeE. Alternately, a combination of any two or more of the botulinumserotypes A-G can be locally administered to control the onset andduration of the desired therapeutic result Furthermore, non-neurotoxincompounds can be administered prior to, concurrently with or subsequentto administration of the neurotoxin to proved adjunct effect such asenhanced or a more rapid onset of denervation before the neurotoxin,such as a botulinum toxin, begins to exert its therapeutic effect.

My invention also includes within its scope the use of a neurotoxin,such as a botulinum toxin, in the preparation of a medicament for thetreatment of a thyroid disorder by local administration of theneurotoxin.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

I claim:
 1. A method for treating hypocalcemia, the method comprisingthe step of direct administration of a botulinum toxin to a sympatheticganglion which innervates the thyroid of a patient, thereby treatinghypocalcemia.
 2. The method of claim 1, wherein the botulinum toxin isadministered in an amount of between 10⁻³ U/kg of patient weight and 35U/kg of patient weight.
 3. The method of claim 1, wherein the botulinumtoxin is made by a Clostridial bacterium.
 4. The method of claim 1,wherein the botulinum toxin is selected from the group consisting ofbotulinum toxin types A, B, C₁, D, E, F and G.
 5. The method of claim 1,wherein the botulinum toxin is botulinum toxin type A.
 6. A method fortreating a hypocalcemia, the method comprising the step ofadministration of a therapeutically effective amount of a botulinumtoxin to a patient, thereby treating hypocalcemia.
 7. The method ofclaim 6, wherein the botulinum toxin is selected from the groupconsisting of botulinum toxin types A, B, C₁, D, E, F and G.
 8. A methodfor treating hypocalcemia, the method comprising the step of localadministration to a sympathetic ganglion which innervates a thyroid Ccell of a therapeutically effective amount of a botulinum toxin, therebydecreasing an excessive calcitonin secretion from the thyroid C cell andtreating hypocalcemia.
 9. The method of claim 8, wherein the botulinumtoxin is selected from the group consisting of botulinum toxin types A,B, C₁, D, E, F and G.